An organism's ability to sense and respond to changes in the energy status of the cell is critical for adapting to fluctuations in the environment throughout lifespan. The AMP-activated protein kinase (AMPK) is activated by low energy levels and plays a crucial role in longevity in invertebrates. While the role of AMPK in mammalian longevity is not yet known, emerging evidence indicates that AMPK is activated in response to dietary restriction and plays a role to prevent age-dependent diseases such as diabetes and cancer. Interestingly, AMPK influences lifespan via both Foxo transcription factor-dependent and -independent mechanisms. Foxo transcription factors are known to regulate lifespan in a variety of species. Our lab has recently shown that AMPK phosphorylates both worm and mammalian Foxo transcription factors. In mammalian cells, AMPK phosphorylation of the FoxoS transcription factor modulates the expression of target genes involved in stress resistance and energy metabolism. These findings raise the possibility that AMPK may regulate genes involved in longevity by directly phosphorylating specific transcription factors. To explore the molecular mechanisms by which AMPK affects gene expression in response to changes in energy levels, I propose the following specific aims: Aim 1: To characterize the role of the phosphorylation of the FoxoS transcription factor by AMPK Aim 2: To identify novel substrates of AMPK that regulate gene expression To achieve these specific aims, I will use a chemical genetics technique in which AMPK has been engineered to specifically label the substrates of AMPK. I will analyze the effects of phosphorylation on AMPK substrates using a combination of molecular and biochemical approaches. Deciphering the mechanisms by which AMPK regulates transcription factors in response to low nutrient levels will increase our knowledge of the signaling pathways that control gene expression programs affecting stress resistance and energy metabolism. As the response to low nutrients is thought to play a key role in longevity induced by dietary restriction, these studies should give key insights into the pathways that influence mammalian aging in response to environmental changes.